The principal objective of this project is to elucidate the structure of the HIV-1 integrase protein, complexed with DNA and/or inhibitors, to use the structural knowledge thus obtained to design better inhibitors of this enzyme with the goal of developing new anti-HIV drugs, and to apply any other computer-aided drug design method that may be helpful in identifying new, promising HIV-1 integrase inhibitors. HIV integrase (IN) is the virally encoded enzyme responsible for integration of the retroviral DNA into the host genome. This step in the life cycle of HIV is essential for viral replication. Inhibition of integration is seen as an attractive target in the development of anti-AIDS therapies because no cellular homologue to IN is known, thus raising the hope that effective anti-IN based drugs with low-toxicity can be developed. The emergence of multidrug-resistant virus phenotypes during administration of cocktails of protease and reverse transcriptase (RT) inhibitors has further highlighted the need for alternative therapeutic approaches. IN is a 32kDa protein that is a product of the gag-pol fusion protein precursor contained in the virus particle. Upon completion of proviral DNA synthesis by RT, IN cleaves two nucleotides from each viral DNA end (3'-processing). After subsequent migration to the host cell's nucleus, IN catalyzes the insertion of the recessed 3'-terminus, generated during the 3'-processing step, into one strand of the host DNA. This reaction is termed 3' end joining (also known as integration or strand transfer) and occurs for both ends of the viral DNA simultaneously. The subsequent gap-joining is presumed to be performed by cellular DNA repair enzymes to yield a fully integrated proviral DNA. Previous work, mainly based on 3D-pharmacophore searches in the NCI database, had yielded a number of inhibitors of IN. With the advent of more, and better, experimental structures (by X-ray crystallography and NMR) of HIV-1 IN as well as of closely related enzymes such as ASV integrase, it has become possible to model larger structures including multimeric models of the full-length protein, for which experimental structures are not available as of yet. We have generated such structures by means of molecular modeling techniques using all available experimental evidence. Special emphasis was placed on obtaining a model of the enzyme's active site with the viral DNA apposed to it as it might be after 3'-processing but before strand transfer, as described in Karki et al., 2004. This model is useful for structure-based inhibitor design of inhibitors which retain activity in vivo. We have made use of these structural models to study the potential binding modes of various diketo-acid HIV-1 IN inhibitors for which no experimental complexed structures are available. The results indicate that the diketo-acid IN inhibitors probably chelate the metal ion in the catalytic site and also prevents the exposure of the 3'-processed end of the viral DNA to human DNA. These models were success fully used for inhibitor development, utilizing resources including those described in our database project, in particular through in silico screening of a database of more than 26 million purchasable screening samples. Current efforts have been focusing on ligand-based inhibitor design, making use of the structural information coming from those few molecules that have made it into late-phase clinical trials or been approved as anti-HIV drug. Based on these structural motifs, a series of novel compounds not covered by IN-related patents were designed and submitted for quotation for synthesis via the newly implemented Semi-Custom Online Synthesis Request System (SCSORS) mentioned in the Database project. From the more than 8,000 compounds quoted by more than 10 different suppliers world-wide, a set of nearly 100 was chosen and submitted for purchase. About 30 compounds were obtained from this set. Some of them exhibited moderate anti-IN activity. Based on these compounds and additional QSAR models as well as structure-based activity predictions, we designed a set of about 2000 possible novel analogs, a subset of which was synthesized by the original supplier identified through SCSORS. A recent extension of this project has been our work on HIV microbicides supported by Intramural-to-Russia Program award funds. The goal of this project is to develop novel HIV microbicides for preventive topical application such as in vaginal gels. While microbicidal activity need not be (solely) based on anti-IN activity, our current efforts are based on a combination of molecular targets including integrase. The other currently used target are reverse transcriptase (RT) and protease (PR). Both ligand-based (SAR/QSAR) inhibitor design approaches and structure-based approaches (docking) have been applied in this project. Several different types of cell-based and ex vivo assays of 48 compounds identified in our current CADD efforts have been conducted. Four compounds with interesting activities were identified. Elaboration of these hits by additional computer-aided drug design approaches and subsequent synthesis as well as additional purchases have been completed. The compilation of all these compounds, for a total of nearly 240 samples, have been assayed in a battery of tests, comprising two cell-based and three enzymatic assays. For 28 of the most-interesting hits, cell-based dose-response assays with six concentrations performed. The two most interesting ones among the nine active compounds found are currently being investigated as potential future lead compounds. The project Exploration of Chemical-Biological Space via a Very Large Database of Synthesizable Compounds to Discover Novel Anti-HIV Agents aims at developing a workflow for efficient retrieval from the SAVI database of compounds with complex predicted desirable ADMET characteristics to create a SAVI-ADMET subset; collection, analysis, curation, and usage of data (including protein expression, targets, and molecular mechanisms) for building of (Q)SAR models from publicly and commercially available databases about the main mechanisms of pathogenesis of HIV/AIDS and HIV-associated comorbidities (on the Russian side); identifying with these (Q)SAR models in SAVI-ADMET single- and multi-target ligands active against HIV-1 and against different types of HIV-related comorbidities; synthesis and biological testing of 50-100 selected compounds either by Russian or U.S. collaborators.